This invention relates to a medicine for humans, and particularly to a pharmaceutical combination of Epidermal Growth Factor (EGF) and Growth Hormone secretagogue hexapeptide (GHRP) for use in preventing tissue damage due to blood flow suppression and in enhancing tissue repair following ischemic damage.
All the organs in the animal body are susceptible to lethal irreversible tissue damage following partial or full-arterial blood flow deprivation, or venous drainage failure. In these scenarios, cellular death is the aftermath of a progressive cascade of pathophysiological changes, which may eventually threaten appropriate multi-organ functioning and an individual's survival.
Exaggerated ROS generation is a key pathological consequence of a myriad of processes linked to tissue hypoperfusion, ischemia/reperfusion, and inflammation (peritonitis, pancreatitis, etc). Tissue hypoperfusion and reactive oxygen species (ROS) over-production are also associated to major surgery, revascularization surgery, extensive burns and multiple traumas. Membranes lipid peroxidation by ROS attack is responsible for cellular demise in many pathologic conditions (T. D. Lucas and I. L. Szweda. Cardiac reperfusion injury. Aging, lipid peroxidation and mitochondrial dysfunction. Proc Natl Acad Sci USA 1998, 95 (2): 510-514).
Depletion of cellular ATP stores is the most acute and threatening consequence of ischemia (Burns T A, Davies R D, McLaren J A, Cerundolo L, Morris J P, Fuggle V S. Apoptosis in ischemia/reperfusion injury of human renal allografts. Transplantation. 1998, 66 (7): 872-876). Along the ischemic process, ATP stores are degraded to hypoxantine and xantine, both acting as substrates for the enzyme xantine oxidase (XO). The large availability of incoming molecular oxygen during the reperfusion period leads to purine oxidation via XO activation, resulting in superoxide anion and hydrogen peroxide generation (Paller M S, Hoidall J R, Ferris J E. Oxygen free radicals in ischemic acute renal failure in the rat. J Clin Invest 1994, 74: 1156-1164.). During ischemia/reperfusion periods, ROS generation and microvascular failure combine to act as a vicious circle, in which activated endothelial cells and circulating leukocytes recruitment/adhesion further increase territorial tissue perfusion and thus cellular hypoxia (Redl H, Gasser H, Hallstrom S, Schlag G. Radical related cell injury. In Pathobiology of shock, sepsis and organ failure. G. Schlag, H. Redl, editors. Springer-Verlag, Heidelberg. Germany 1993, 92-110); (Ledebur H C, Parks T P. Transcriptional regulation of the intercellular adhesion molecule 1 gene by inflammatory cytokines in human endothelial cells: essential roles of a variant NF-kB site and p65 homodimers. J Biol Chem 1995, 270: 933-943).
ROS may activate NF-kB in tissues infiltrated by inflammatory cells (Conner E M, Brand S J, Davis J M, Kang D Y, Grisham M B. Role of reactive metabolites of oxygen and nitrogen in inflammatory bowel disease: toxins, mediators, and modulators of gene expression. Inflamm Bowel Dis 1996, 2: 133-147), whereas the myeloperoxidase (MPO) enzyme system is activated in polymorphonuclear cells infiltrating hypoxic tissue which further amplify the tissue damage cascade (Kurose I, Argenbright L W, Wolf R, Lianxi L, Granger DN. Ischemia/reperfusion-induced microvascular dysfunction: role of oxidants and lipid mediators. Am J Physiol 1997, 272: H2976-H2982). In this hostile environment, local thrombogenic mechanisms are activated which results in capillary plugging and hypoxia territorial expansion. As a consequence of this cascade, cellular death ensues by necrosis and/or apoptosis, which may compromise an organ's viability (Tredger M J. Ischemia-reperfusion injury of the liver: treatment in theory and practice. Biofactors 1998, 8 (1-2): 161-164). Endothelial/inflammatory cell reactivity renders a large number of chemical soluble mediators such as nitric oxide, pro-inflammatory cytokines, pro-coagulant and vasoactive agents which may trigger the Systemic Inflammatory Response Syndrome (SIRS) if the body is incapable of counteracting its immune dissonance (Kowal-Vern A, McGill V, Gamelli R L. Ischemic necrotic bowel disease in thermal injury. Archives of Surgery 1997, 132 (4): 440-443). Major burns are medical emergencies demanding intensive and multiple medical efforts to save a patient's life. In burn patients, gut hypoperfusion 1 ischemia seems to play a critical role in orchestrating the SRIS (Wang P, Ba Z F, Cioffi W G, Bland K I, and Chaudry I H. Is gut the “motor” for producing hepatocellular dysfunction after trauma and hemorrhagic shock? Journal of Surgical Research 1998, 74: 141-148). Intestinal barrier failure is of paramount clinical relevance as the gut epithelium acts as a frontier between a septic/toxic lumen and a sterile internal environment (Sheridan R L, Ryan C M, Yin L M, Hurley J, Tompkins R G. Death in the burn unit. Sterile multiple organ failure. Burns 1998, 24 (4): 307-311).
In this regard, both experimental and clinical findings converge to show the importance of an adequate intestinal perfusion during systemic stress as to preserve barrier integrity (Tabata T, de Serres S, Meyer A A. Differences in IgM synthesis to gut bacterial peptidoglycan ploysaccharide after burn injury and gut ischemia. Journal of Burn Care and Rehabilitation 1996, 17 (3): 231-236). Furthermore, recent evidences also indicate that intestinal tissue acts as a pro-inflammatory cytokine-generating source when intestinal-associated lymphoid tissue is activated by ischemia. Multiple organ failure (MOF) is a first cause of death in patients admitted under intensive care conditions, and is the most frequent complication for burn victims, involving up to a 70% of patients in highly specialized burn treatment units.
In order to attenuate the consequences of the ischemia/reperfusion process in certain organs, a large number of synthetic or natural compounds have been pre-clinically or clinically examined. For the case of intestinal ischemia, angiotensin II inhibitors were experimentally evaluated (Tadros T, Taber D L, Heggers J P, Herndon D N. Angiotensin II inhibitor DuP753 attenuates burn and endotoxin-induced gut ischemia, lipid peroxidation, mucosal permeability and bacterial translocation. Ann Surg 2000; 231: 566-576). Platelet activating factor inhibitors (Sun Z, Wang X, Deng X, Lasson A, Soltesz V, Borjesson A, Andersson R Beneficial effects of lexipafant, a PAF antagonist on gut barrier dysfunction caused by intestinal ischemia and reperfusion in rats. Dig Surg 2000; 17: 57-65), and enhancers of nitric oxide release were also studied (Ward D T, Lawson S A, Gallagher C M, Conner W C, Shea-Donohue D T. Sustained nitric oxide production via L-arginine administration ameliorates effects of intestinal ischemia-reperfusion. J Surg Res 2000; 89: 13-19). Other approaches include anti-oxidant therapy such as allopurinol alone or in combination with vitamins C and E (Kacmaz M, Otzurk H S, Karaayvaz M, Guven C, Durak I. Enzymatic antioxidant defense mechanism in rat intestinal tissue is changed after ischemia-reperfusion. Effects of allopurinol plus antioxidant combination. Can J Surg 1999; 42: 427-431).
Despite this, efforts forwarded to spark cellular natural defensive mechanisms are scarce (Pialli S B, Hinmn C E, Luquette M H, Nowicki P T, Besner G E. Heparin-binding epidermal growth factor-like growth factor protects rat intestine from ischemia/reperfusion injury. J Surg Res1999; 87: 225-231). Renal demise following ischemia/reperfusion has fueled the search for nephroprotective agents, including the generation of the so-called lazaroids, which have shown to confer global protection to the ischemic kidney (De Vecchi E, Lubalti L, Beretta C, Ferrero S, Rinaldi P, Galli K M, Trazzi R, Paroni R. Protection from renal ischemia-reperfusion injury by the 2-methylaminochroman U83836. Kidney Int 1998, 54: 857-863). Other studies document the salutary effects of teofilin in renal protection, chiefly as an antagonist to adenosine receptors (Jenik A G, Ceriani J M, Gorenstein A, Ramirez J A, Vain N, Armadans M, Ferraris J R. Randomized, double-blind, placebo-controlled trial of the effect of theophylline on renal function in term neonates with perinatal asphyxia. Pediatrics 2000; 105: E45).
Administration of the atrial natriuretic peptide (Auriculin) did not reduce mortality in patients affected by acute renal failure and remote organs complications were not reduced (Weisberg L S, Allgren R L, Genter F C, Kurnik B R. Cause of acute tubular necrosis affects its prognosis. The Auriculin Anaritide Acute Renal Failure Study Group. Arch Intern Med 1997; 157: 1833-1839). Nephroprotection has been attributed to the enzyme superoxide dismutase (SOD) when injected at high dose levels in patients undergoing renal transplant surgery (Schneeberger H, Schleibner S, Illner W D, Messmer K, Iand W. The impact of free-radical mediated reperfusion injury on acute and chronic rejection events following cadaveric renal transplantation. Clin Transpl 1993; 219-232).
The benefits of EGF and TGF-alpha in ameliorating toxic and ischemic acute renal failure are shown in U.S. Pat. No. 5,360,790. Although the parenteral administration of some growth factors exhibiting nephroprotective effects has proven effective in experimental models, so far clinical results are discouraging. A controlled multicenter clinical trial did not show the expected benefits of IGF-I in acute renal failure patients when compared to placebo counterparts (Hirschberg R, Kopple J, Lipsett P, Benjamin E, Minei J, Albertson T, Munger M, Meztler M, Zaloga G, Murray M, Lowry S, et al. Multicenter clinical trial of recombinant human insulin-like growth factor I in patients with acute renal failure. Kidney Int 1999; 55:2423-2432). In a further clinical trial employing IGF-I for acute renal failure, the lack of effect was confirmed (Kopple J D, Hirschberg R, Guler H P, Pike M, and Chiron Study Group: lack of effect of recombinant human insulin-like growth factor-1 (IGF-1) in patients with acute renal failure (ARF). J Am Soc Nephrol 1996; 7: 1375). Scarce progress in organ preservation technology achieved thus far remains the most important limitation of new organ availability for transplantation. Furthermore, ex-vivo preservation agents have yielded conflicting effects (Schlumpf-R; Candinas-D; Weber-M; Rothlin-M; Largiader-F. Preservation of kidney transplants with a modified UW solution initial clinical results. Swiss-Surg. 1995(4): 175-80; discussion 180-1); and organ biochemical and functional deterioration following implantation into the recipient remains the first cause of non-immune rejection (Barber E, Menéndez S, León O S, Barber M O, Merino N, Calunga J L, Cruz E, and Bocci V. Prevention of renal injury after induction of ozone tolerance in rats submitted to warm ischemia. Mediators of Inflammation 1999; 8: 37-41). An untoward effect reported for some preservation agents is its interference in platelet aggregation mechanism, thus leading to profuse bleeding (Salat A, Mueller M R, Boehm D, Stangl P, Pulaki S, Laengle F. Influence of UW solution on in vitro platelet aggregability Transpl-Int. 1996; 9 Suppl 1: S429-431). Vasospasm and thrombosis in the post-reperfused organ are amongst the inconveniences reported (Jeng-LB; Lin-PJ; Yao-PC; Chen-MF; Tsai-KT; Chang-CH. Impaired endothelium-dependent relaxation of human hepatic arteries after preservation with the University of Wisconsin solution. Arch-Surg. 1997 January; 132(1): 7-12). The medical community still expects more efficient and less expensive organ preservation solutions (Rentsch M, Post S, Palma P, Gonzalez A P, Menger M D, Messmer K. Intravital studies on beneficial effects of warm Ringer's lactate rinse in liver transplantation. Transpl Int. 1996; 9(5): 461-7). The failure of IGF-I in affording an efficient nephroprotective effect in the clinical arena has introduced the notion that therapy with a single growth factor is not sufficient to stimulate cellular survival during ischemia/reperfusion, and that growth factor cocktails will be more efficacious (Playford R J. Peptides and gastrointestinal mucosa integrity. Gut 1995, 37: 595-597).
The salutary effects of Epidermal Growth Factor (EGF) in protecting organ damage during ischemia/reperfusion episodes was claimed by European patent EP 0 357 240 B1. However, cerebral protection is only achieved with very high EGF concentrations (1 mg/kg). A lower dose of 0.1 mg/kg only showed a modest protective effect despite the dose being high for a substance like a growth factor. These facts impose limitations to the invention, the first one is related to the high cost of the treatment as repeated injections (4 to 5) are required to achieve an effect in the animal. As an example, a 70-kg human subject would require 70 mg of EGF in a single injection, which will have to be periodically repeated to ensure a clinical effect. The second limitation is pharmacologic. The examples shown in the patent suggest that there is a very narrow therapeutic window that hinders the possibility of establishing an Effective Dose 50 (ED50) and a dose-response curve. A third limitation is associated with high EGF doses. Reports exist demonstrating that in rats and monkeys, EGF may depress heart output and arterial pressure (Keiser J A, Ryan N J. Hemodynamic effect of EGF in conscious rats and monkeys. PNAS USA 1996; 93(10): 4957-4961). Normal cell cycle progression may also be perturbed by high concentrations of EGF (Bennett N T, Schultz G S. Growth factors and wound healing: Biochemical properties of growth factors and their receptors. Am J Surg 1993, 165:728-737). The potential benefits of EGF intervention in protecting the liver and the intestines against ischemia/reperfusion seem to require further elucidation. Mounting evidence indicates that the parenteral administration of EGF seem to confer protection to a variety of internal epithelial organs following acute blood flow suppression. In experimental conditions with animals exposed to a chemical, which blocks ATP synthesis or increases the rate for EROs generation, EGF intervention proved to be useful by reducing organ damage. Furthermore, repeated EGF administration assists in enhancing tissue regeneration, adaptation and functionality. All of these benefits of EGF therapy may only be achieved under repeated administration regimes and high concentrations of the polypeptide. Often, these benefits are modest, which further strengthen the notion that a combined therapy of growth factors is rather preferable. In the context of ischemia, EGF seems to attenuate tissue damage if ischemia time is less than 60 minutes. For larger ischemia periods, EGF therapy is worthless. This is an obvious limitation for EGF therapy as protection for larger ischemic periods is required in surgical practice.
Although the need of growth factor combination has been emphatically claimed for a large period of time, there is no combination available in the clinical armamentarium.